RNA viruses whose genome is composed of a single RNA strand capable of replication in the cytoplasm of a host by direct RNA replication are widespread, many varieties of which are known and which infect animals, plants and bacteria. Such viruses are sometimes termed "(+) strand RNA viruses" since the infective RNA strand, that normally found encapsidated in the virus particle, is a messenger-sense strand, capable of being directly translated, and also capable of being replicated under the proper conditions by a direct process of RNA replication. Viruses belonging to this group include, but are not limited to, the picornaviruses, the RNA bacteriophages, the comoviruses, and various single component and multicomponent RNA viruses of plants. A partial listing of such viruses would include polio virus, sindbis virus, Q.beta. bacteriophage, tobacco mosaic virus, barley stripe mosaic virus, cow pea mosaic virus, cucumber mosaic virus, alfalfa mosaic virus and brome mosaic virus. In some cases, the entire virus genome is contained within a single RNA molecule, while in other cases, most notably the multicomponent RNA plant viruses, the total genome of the virus consists of two or more distinct RNA segments, each separately encapsidated. (For general review, see General Virology, S. Luria and J. Darnell; Plant Virology 2nd ed., R. E. F. Matthews, Academic Press (1981); and for a general review of (+) strand RNA replication, see Davies and Hull (1982) J. Gen. Virol. 61,1). Within the group there are wide variations in capsid morphology, coat proteins, genetic organization and genome size.
Despite the well-documented diversity, recent studies have shown striking similarities between the proteins which function in RNA replication. Sequence homologies have been reported between the cowpea mosaic virus, poliovirus and foot-and-mouth disease virus (Franssen, H. (1984) EMBO Journal 3,855), between non-structural proteins encoded by alfalfa mosaic virus, brome mosaic virus and tobacco mosaic virus, Haseloff, J. et al. (1984), Proc. Nat. Acad. Sci. USA 81, 4358, and between the same proteins and proteins encoded by sindbis virus, Ahlquist, P. et al. (1985) J. Virol. 53, 536. Evidence of such substantial homology in proteins related to the replication functions indicate that the viruses share mechanistic similarities in their replication strategies and may actually be evolutionarily related. In the present invention, modifications to the genomic RNA of a (+) strand RNA virus are disclosed. The modified RNA is used to transfer a desired RNA segment into a targeted host cell and to replicate that segment and express its function within the host cell. A virus known to be representative of the common replication functions of (+) strand RNA viruses was chosen to exemplify the present invention herein.
Brome mosaic virus (BMV) is one member of a class of plant viruses characterized by a multipartite RNA genome. The genetic material of the virus is RNA, and the total genetic information required for replication and productive infection is divided into more than one discrete RNA molecule. The class, termed multipartite RNA viruses herein, includes, besides BMV, such viruses as alfalfa mosaic virus (AMV), barley stripe mosaic virus, cowpea mosaic virus, cucumber mosaic virus, and many others. Virus particles are generally composed of RNA encapsidated by a protein coat. The separate RNA molecules which comprise the total genome of a given multipartite virus are encapsidated in separate virus particles, each of which has the same protein composition. Infection of a host plant cell occurs when a virus particle containing each of the RNA components of the viral genome has infected the cell, for example by exposing a plant to a virus preparation containing a mixture of all necessary viral components. Infection may also be achieved by exposing a plant cell or protoplast to a mixture of the RNA components. A subclass of the multipartite RNA viruses (termed subclass I herein) requires coat protein in addition to viral RNA for replication and productive infection. AMV is an example of a subclass I multipartite virus. Another subclass (termed subclass II herein) does not require coat protein, the component RNAs being both necessary and sufficient for replication and productive infection. BMV belongs to subclass II. The BMV genome is divided among three messenger-sense RNAs of 3.2, 2.8 and 2.1 kilobases (Ahlquist, P. et al. (1981) J. Mol. Biol. 153,23; Ahlquist, P., et al. (1984) J. Mol. Biol. 172,369). The term "messenger-sense" denotes that the viral RNAs can be directly translated to yield viral proteins, without the need for an intervening transcription step.
Complete cDNA copies of each of the three BMV genetic components have been cloned in a general transcription vector, pPM1, described by Ahlquist, P. and Janda, M. (1984) Mol. Cell Biol. 4,2876. Three plasmids have been selected, pB1PM18, pB2PM25 and pB3PM1 containing, respectively, cDNA copies of BMV-RNA1, BMV-RNA2 and BMV-RNA3. The three plasmids constitute, as a set, the complete BMV genome.
DNA from each of the three BMV cDNA-containing plasmids can be cleaved at a unique EcoRI site. The linear DNA thus produced can be transcribed in vitro in a reaction catalyzed by RNA polymerase. A modified .lambda. P.sub.R promoter in the transcription vector, pPM1, allows RNA synthesis to initiate exactly at the 5' terminus of each BMV sequence, and transcription continues to the end of the DNA template, adding 6-7 nonviral nucleotides at the 3' ends of the transcripts. When transcription is carried out in the presence of a synthetic cap structure, m.sup.7 GpppG, as described by Contreras, R., et al. (1982) Nucleic Acids Res. 10,6353, RNA transcripts are produced with the same capped 5' ends as authentic BMV RNAs. These RNAs are active messengers in in vitro translation systems and direct production of proteins with the same electrophoretic mobilities as those translated from authentic BMV RNAs.